Many industrial, scientific and medical processes involve the measurement of the chemical composition of tissue of the human body for a variety of applications. In most cases the accuracy of the measurements is of great importance for the quality of the output of the process mentioned. A specific type of tissue measurements involve the measurement of the transparent media of the eye, as is applied, for example, in ophthalmology where proteins, pharmacological substances, or other molecules in aqueous humor are determined, wherein the presence of these substances provide an indication of the quality of the eye and sometimes information concerning underlying diseases.
Most methods of measuring the pathologic conditions in the eye are based on chemical and histological techniques to acquire information on molecule changes in the tissue or identification of pathogens which implies the use of spectroscopic techniques like absorption, scattering, electronic spin, and/or mass spectroscopy.
The limitations of these methods are that the most of them are performed in vitro, which makes invasive procedures in the eye necessary that may led to complications like hypotony or an endophthalmitus of the eye and is a frightening intervention from the patients point of view.
A solution is the application of in vivo spectroscopy, especially with Raman spectroscopy. Raman spectroscopy offers an opportunity to detect molecules qualitative as well quantitative and is a valuable tool to investigate biological materials in aqueous environments. For Raman spectroscopy in the living eye a laser beam is focussed in the tissue of interest and the back scattered light comprising the Raman signals is gathered, e.g. in a confocal device, and analyzed in a spectrometer.
The limitation of these methods is the restriction of the amount of incident light due to the vulnerably of the retinal tissue for light exposure, making the detection of molecules that are present in low concentration difficult if not impossible.
A solution is the application of an oblique illumination with respect to the visual axis of the eye in such a way that the incident excitation beam is not aimed at the retinal tissue. An oblique illumination of the eye can be performed with a device according to the preamble above.
A disadvantage of this technique is that the oblique alignment with the corneal surface interferes with the optical quality of the setup in such a way that it obstructs the confocal tissue selection.